The effect of KCNQ5 channel intervention on the development of form deprivation myopia in guinea pigs

The effect of KCNQ5 channel intervention on the development of form deprivation myopia in guinea pigs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The effect of KCNQ5 channel intervention on the development of form deprivation myopia in guinea pigs Qin Yang, Jiahao Niu, Yuling Tang, Yuanyuan Hu, Guimei Zhou, Huilan Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6127485/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: To investigate the effect of KCNQ5 on myopia and explore the mechanism by which potassium channel influence myopia in vivo. Methods: Animals were divided into five groups: normal control, form deprivation (FD), solvent control, FD + Retigabine and FD + XE991. The concentrations of the Retigabine administered were 100μM and 500μM, while those for XE991 were 20μM, 50μM and 100μM. Injections and biometric examinations were performed at predetermined intervals. The expression levels were evaluated using RT-PCR and Western Blot, and cell apoptosis was detected via TUNEL. Results: After four weeks, the progression of refraction and axial length in FD + Retigabine were significantly slowed down but accelerated in FD + XE991 compared to the FD. Statistically significant differences were observed between 50μM and 100μM. There was no significant difference in IOP among groups. The mRNA and protein levels of KCNQ5 increased in FD + Retigabine but decreased significantly in doses of XE991 treatment when compared with FD controls. TUNEL staining indicated no significant differences in positive cell rates across all retinal layers. Conclusions: Intervention targeting KCNQ5 channels appears to modulate the development of FD myopia, suggesting that potassium channel may play a role in underlying mechanisms associated with myopia. Biological sciences/Biochemistry Biological sciences/Physiology Health sciences/Health care Health sciences/Pathogenesis KCNQ5 Potassium channel Retigabine XE991 intravitreal injection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Myopia is the most common refractive error in clinical practice, which affects visual acuity and visual quality and even harms visual function. It is the main cause of visual impairment and the major public health problem worldwide. 1 Moreover, myopia among children and adolescents has become increasingly serious in recent years. However, at present, the pathogenesis of myopia has not been clarified, resulting in limited means of correction and a lack of effective intervention measures in clinic. Therefore, it is scientific and clinical significance to reveal the mechanism of myopia. Recent studies have shown that potassium ion (K + ) homeostasis is an important manifestation of myopia development, in which various related channels regulating potassium transport in ocular tissue may play a key role. 2 , 3 Our previous study revealed that the concentration of K + increased, while the expression of KCNQ5 was significantly down-regulated in the guinea pig retina of form-deprivation myopia (FDM). Studies have also found that KCNQ5 is expressed in bovine RPE and is involved in controlling the concentration of K + in the retina, which plays an important role in maintaining the physiological functions of photoreceptors, Müller cells and RPE cells. 4 Besides, we verified that the intervention effects of activating and inhibiting KCNQ5 channels on retinal muscarine (M) currents in guinea pigs through a series of in vitro experiments. 5 These findings suggest that retinal KCNQ5 potassium channel may be involved in the occurrence and development of myopia. The present study aimed to observe the effect of KCNQ5 channel intervention on the development of FDM in guinea pigs through in vivo experiment, and to further investigate the role of KCNQ5 channel in myopia. MATERIALS AND METHODS Animals and Study Design A total of 85 pigmented male guinea pigs aged three-week-old (120–150 g) were supplied by Chongqing Tengxin Biotechnology Co., Ltd., The animals were raised at 25 ± 2℃ and in a 12-h light-dark cycle with approximately 300–500 lux. 6 Monocular FDM was induced by translucent facemasks made of latex balloons, the right eye was covered for a period of 4 weeks. All animal experiments were approved by the Animal Care and Ethics Committee of North Sichuan Medical College (NSMC 202116), and followed the Statement of Association for Research in Vision and Ophthalmology (ARVO) for the Use of Animals in Ophthalmic and Visual Research. All experimental procedures and related operations in this study were carried out in accordance with the standards of ARRIVE guidelines. The animals were randomly divided into 5 groups: control group (NC) with untreated bilateral eye, FD group with unilateral FDM, solvent group (FD + DMSO) with unilateral FDM and intraocular injection of a solvent containing 10% DMSO into the covered eye. Besides, on the basis of FD, retigabine group (FD + Retigabine) was injected with different concentrations of Retigabine, and XE991 group (FD + XE991) was injected with different concentrations of XE991. The concentrations of Retigabine were 100µM and 500µM, and XE991 were 20µM, 50µM and 100µM, respectively. The animals were then subdivided into 8 subgroups according to the above treatments and drug concentration differences (Table 1 ). Table 1 The design of experimental grouping Group Treatment Drug concentration Ⅰ NC 0 Ⅱ FD 0 Ⅲ FD + DMSO 10% DMSO Ⅳ FD + 100 RTG 100µM Retigabine + 10% DMSO Ⅴ FD + 500 RTG 500µM Retigabine + 10% DMSO Ⅵ FD + 20 XE991 20µM XE991 + 10% DMSO Ⅶ FD + 50 XE991 50µM XE991 + 10% DMSO Ⅷ FD + 100 XE991 100µM XE991 + 10% DMSO Drugs and Intravitreal injection Drugs Retigabine (selleckchem, United States) and XE991 (Sigma, United States) were dissolved in DMSO (Sigma, United States), and the solvent was prepared from 0.9% saline solution containing 10%DMSO. The drug stock solution was diluted with solvent before using to ensure that the DMSO content in each animal was maintained at 10%. Before injection, the diluted drugs were separately packed and stored in sterile EP tubes at 4℃. Injections were performed once every 3 days at a fixed time with the injection volume of 10µl. Iodophor was used to sterilize around the eyes, and aubucaine hydrochloride (Santen, Japan) were used for surface anesthesia. Oxyfloxacin ophthalmic ointment (Santen, Japan) was used twice daily to prevent infection. After four weeks, animals were euthanized and the ocular tissue was taken for follow-up studies. 7 Measurement of Ocular Biometric Parameters The ocular biometric parameters were measured at five time points (0, 1, 2, 3, and 4 weeks of FD). Refraction was recorded by an independent experienced optometrist using retinoscopy (66 Vision-Tech Co., Ltd., China), and analyzed using the spherical equivalent (SE, SE = spherical power + 1/2 cylindrical power). 8 Guinea pigs were given cycloplegic drops (compound tropicamide; Santen Pharmaceutical Co., Ltd., Japan) at 5-min intervals for three times to dilate pupils and inhibit ocular accommodation. The anterior chamber depth, lens thickness, and vitreous cavity length (the sum of the three parameters was AL) (Zhang et al., 2018) were measured with an A-ultrasound (Cinescan, Quantel Medical, France) with an ultrasound probe frequency of 11 MHz, and the conduction velocities were set as 1557.5, 1723.3, and 1540 m/s, respectively. Before measurement, topical anesthesia was performed with oxybucaine hydrochloride (Santen Pharmaceutical Co., Ltd., Japan), and then the probe tip was perpendicular to the center of the cornea. Each measurement comprised an average of at least ten scans. 9 Intraocular pressure (IOP) was measured using a tonometer (iCare, Finland) during the same period after each intravitreal injection. The animals were measured under anaesthesia, and each eye was measured 5 times to take an average value for recording. 10 Tissue preparation At the end of 4-week treatment, the guinea pigs were anesthetized with 3% sodium pentobarbital (50 mg/kg) until the cornea and pain reflex disappeared. The eyeballs were enucleated and positioned in a centrifuge tube and fixed in 4% paraformaldehyde for TUNEL staining. Additional eyeballs were enucleated, then the cornea, lens, and vitreous were quickly removed on ice. The retinas were snapped frozen in liquid nitrogen, and stored at − 80◦C. 5 Real-time PCR Total RNA was respectively extracted from the retinas using trizol reagent (Beyotime, Biotechnology, China) according to the manufacturer’s protocol. This was followed by reverse-transcription of 1 µg of total RNA to cDNA using Takara reverse transcriptase and the random primer provided in the kit (Takara, Japan). Real-time PCR was performed on a Roche LightCycler instrument with Takara SYBR green II kit using resultant cDNA as template. The primer sequences in this study were as follows: KCNQ5 forward primer 5'-ACTTGGCTGGGAAGGTTGCTTTC-3' and reverse primer 5'-GTGTTTCTGGCGGTGTTGTTCTTG-3’, β-actin forward primer 5'-CTGGGTATGGAATCCTGTGGCATC-3' and reverse primer 5'-CAGCACTGTGTTGGCATAGAGGTC-3'. The amplification was performed under the following cycling conditions: The first step was an initial denaturation at 95◦C for 30 s, followed by 40 cycles of denaturation at 95◦C for 10 s, annealing at 59◦C for 60 s, and extension at 97◦C for 1 s. At the end of the amplification, calculating the relative expression of target genes in each group with the 2^ −△△Ct method. 2 , 11 Western blotting The RIPA lysis buffer (Beyotime Biotechnology, China) containing 1mM PMSF was added to the retina for full cracking to extract proteins from tissues. This was followed by the centrifugation at 12,000 rpm/min for 10 min to collect the supernatants for western blot analysis. Protein concentration was determined by the BCA kit (Beyotime Biotechnology, China). The samples were mixed with a 5 × loading buffer (Beyotime Biotechnology, China) and then placed in 70◦C for 15 min. The protein samples were loaded on 8% SDS-PAGE gels and electrotransferred onto a nitrocellulose membrane. And the membranes were blocked with 5% non-fat milk at room temperature for 2h, the primary antibody of KCNQ5 (1:500, Invitrogen, United States) and β-tubulin (1:5,000, Beyotime Biotechnology, China) were incubated with the membranes at 4◦C overnight. After that, the membranes were incubated with goat anti-rabbit secondary antibody (1:10,000; Boster, China) at room temperature for 1 h. Finally, protein bands were visualized using the FUSION-FX7 imaging system (Vilber Lourmat, France) and the optical density was analyzed using Image J software. 12 TUNEL staining TUNEL staining (Kaiji Biotechnology Co. Ltd., China) was performed to detect apoptotic cells in the retina. The paraffin slices were baked overnight in an oven at 60℃. After dewaxing and rehydrating, the slices were washed 3 times with PBS for 5min each time. It was incubated with protease K solution at 37℃ for 30min (1:50). And then they were washed 3 times with PBS for 5min each time and incubated with TdT enzyme reaction solution at 37℃ for 1h away from light. After washing with PBS for 3 times, Streptavidin-HRP was incubated at 37℃ for 30min in the dark. After 3 washes in PBS, and then dropped with secondary antibody (Boster, China) and incubated at 37◦C for 1 h. The DAB reagent (ZSBg-Bio, China) was subsequently used to detect the immunoactivity. Then the sections were counterstained with hematoxylin (Boster, China), and dehydrated in different concentrations of ethanol. Finally, after the sections were sealed with neutral resin, images were captured using an optical microscope. Image J software was used to measure the optical density of positive retinal cells. 13 Statistical analysis All data were represented as mean ± Standard Deviation (SD), and Statistical software SPSS 25.0 was used for statistical analysis. GraphPad Prism 8.0 and Photoshop were performed for drawing. One-way analysis of variance was used to compare the differences in biological parameters and molecular levels among the groups, multiple comparisons between groups were performed by LSD-t method. All the results shown represent at least three independent experiments, and P < 0.05 was considered statistically significant. RESULTS Effect of drug intervention on refractive status and AL of guinea pigs The results in Fig. 1 showed that after 4 weeks, the refraction in the FD group was about − 3.75 ± 1.99D and the AL was about 8.41 ± 0.11mm. Compared with the FD group, the refraction and AL of the eye in the FD + DMSO group had no statistical significance ( P = 0.79, P = 0.59, respectively). In FD + 100µM Retigabine group and FD + 500µM Retigabine group, both the refraction ( P < 0.05, P < 0.01, respectively) and the AL (all P < 0.01) decreased. On the contrary, in FD + 20µM XE991 group, FD + 50µM XE991 group and FD + 100µM XE991 group, the refraction increased ( P = 0.73, P < 0.05, P < 0.01, respectively) and AL lengthened ( P = 0.82, P < 0.05, P < 0.01, respectively). Effects of drug intervention on intraocular pressure of guinea pig Figure 2 showed the results of IOP in each group after each injection. There was no significant difference in IOP among all groups at each time point of intravitreal injection (all P > 0.05), and the IOP fluctuated between 10-12mmHg in each group. 14 Effect of drug intervention on mRNA level of KCNQ5 in guinea pig Figure 3 showed the mRNA levels of KCNQ5 in retinal tissues of guinea pigs in each group. The mRNA expression level of FD and FD + DMSO groups was lower than the NC group. The expression levels of Retigabine at injection concentrations of 100µM and 500µM were significantly higher than the FD group, and the differences were statistically significant (all P < 0.01). In addition, the expression of KCNQ5 mRNA in the groups injected with XE991 gradually decreased with the increase of drug concentration, and when the concentration increased to 100µM, there was statistical significance in the expression of KCNQ5 mRNA compared with the FD group ( P < 0.05). Effect of drug intervention on protein level of KCNQ5 in guinea pig Figure 4 showed that after the Retigabine was injected into the guinea pigs, the expression of KCNQ5 protein in retina was significantly up-regulated compared with FD group (all P < 0.05). There was no significant difference in the expression of KCNQ5 protein between the FD + DMSO group and the low concentration (20µM) XE991 group, however, the expression of KCNQ5 protein in FD + 50µM XE991 group and FD + 100µM XE991 group was significantly decreased ( P < 0.05). Effect of drug injection on apoptosis of guinea pig retina cells TUNEL staining showed that different degrees of apoptosis could be observed in the retina of NC group, FD group, injection solvent group (FD + DMSO), high concentration Retigabine group (500µM RTG) and high concentration XE991 group (FD + 100µM XE991). Image J software analysis showed that the rate of positive cells in each group was 3.95% ± 0.31% in NC group, 4.73% ± 0.55% in FD group, 4.92% ± 0.95% in FD + DMSO group, 4.81% ± 0.48% in FD + 500µM Retigabine group, and 5.13% ± 1.01% in FD + 100µM XE991 group, and there was no significant difference in TUNEL staining positive cell rate among all groups ( P < 0.05). DISCUSSION K + transport is mainly regulated by K + channels and K + transporters. Previous studies have shown that K + transporters NKCC and Na + /K + -ATPase are involved in the development of myopia. 15 KCNQ5 is one of the important channels that may regulate K + transport between retina and choroid. The differential expression of KCNQ5 in myopic guinea pigs has been confirmed in the previous study, and the expression of KCNQ5 in mRNA and protein levels were lower than the NC group. Meanwhile, elevated K + concentration in the retina of FDM guinea pigs was also observed. 5 These indicating that the KCNQ5 channel may be inhibited during the development of myopia, and preliminarily determined that it may be involved in the regulation mechanism of myopia. KCNQ5 is a member of the KCNQ channel family, which is a voltage-dependent potassium channel, also known as the M channel, because it is sensitive to the regulation of Muscarinic acetylcholine receptors. Retigabine and XE991 act as specific activators and inhibitors of KCNQ channels respectively, and have also been widely used for positive and negative regulation of KCNQ5 channels. 16 , 17 At present, most studies on KCNQ5 channel focus on electrophysiological and neurosystemic diseases, in which the changes of M-type potassium current regulated by KCNQ5 channel were explored. 16 , 18 In the present study, it was found that the injection of activators and inhibitors of KCNQ5 channel into the guinea pig vitreous could regulate the expression of KCNQ5, and had different effect on the refractive state of guinea pigs, which proved that KCNQ5 channel was indeed involved in the development of FDM in guinea pigs. As a crucial regulator, M channels can inhibit neuronal excitability when activated, and vice versa. In fact, studies have found a link between the M channel and the myopia development. For example, atropine, as a Muscarinic acetylcholine receptor nonspecific antagonist, has been used clinically to intervene in myopia and has been shown to be effective. Animal experiments have also confirmed that acetylcholine antagonists, dopamine receptors and other related agents have a certain control effect on the occurrence and development of myopia. 20 However, its side effects such as photophobia and paralytic regulation can be unpleasant, and even lead to cataracts. 21 Therefore, it is still significant to further explore myopia mechanism and potential drugs. Retigabine plays a role in activating KCNQ2-5 channels. As a activator of KCNQ channels, and has been used as a specific activator of KCNQ5 channel in many studies. 16 , 17 Therefore, in this study, Retigabine was selected as an intervention drug of KCNQ5 channel for animal models. At the beginning of the study, it was found Retigabine could affect the development of refraction and AL when the concentration of the drug injected into the guinea pigs was increased to 100µM, and it was slower in FD + Retigabine group than in FD group (Fig. 1 ). RT-PCR and Western Blot results showed that both low (100µM) and high (500µM) concentrations of Retigabine significantly increased the expression of KCNQ5, compared with FD group (Figs. 3 and 4 ). This was consistent with the effect of Retigabine on refractive development. These results indicated that Retigabine can inhibit the development of myopia in guinea pigs by activating the expression of KCNQ5. XE991 is a commonly drug for experimental studies on potassium channel inhibitors. 24 In this study, it was found that intra-abdominal injection of XE991 could cause a higher mortality in guinea pigs, which was speculated to be related to the related nerve response caused by XE991, but no further study was conducted. Therefore, in the follow-up experiment, we selected 3 concentrations as gradients to be administered the drug by intravitreal injection. The results showed that low concentration (20µM) of XE991 slightly inhibited the expression function of KCNQ5, but had no significant effect on refractive development ( P >0.05 ). However, the medium concentration (50µM) and high concentration (100µM) of XE991 can promote the development of refraction and AL of guinea pigs from the early modeling stage, and lead to higher transcription and protein expression levels of KCNQ5 than FD group. These suggested that XE991 as an inhibitor of KCNQ5 channels may play an opposite role in Retigabine, inhibiting the expression of KCNQ5 and promoting the development of FDM in guinea pigs. The administration route in this study was intravitreal injection, and the administration frequency was slightly higher (3 times/day). However, TUNEL staining results showed no statistically significant difference in the rate of positive cells among all groups (Fig. 5 ), indicating that these drugs to intravitreal injection did not cause significant retinal toxicity. Therefore, to a certain extent, the drug concentration and administration method selected in this study were relatively safe. However, there are some limitations in our current study, including that mRNA and protein levels did not show dose-dependent activation and inhibition similar to the results of ocular parameters, possibly due to insufficient precision of drug concentration or differences of drug absorption in vivo. CONCLUSION In conclusion, the present study underscores the significance of retinal KCNQ5 in myopia development in guinea pigs. Furthermore, it established that Retigabine exerts an inhibitory effect on the progression of FDM, while XE991 demonstrates an opposing influence. As previously acknowledged, we hope that our study will shed light on we aspire for our research to illuminate the pathogenesis of myopia and contribute to future prevention and treatment strategies for this condition. Declarations Conflict of interests The authors declare that they have no competing interests. Funding This study was supported by Natural Science Foundation Project of Science & Technology Department of Sichuan Province (No. 2023NSFSC0595), and Key Project of the Affiliated Hospital of North Sichuan Medical College (No. 2023ZD010). Author Contribution QY performed the experiments, analyzed the data, and wrote the original manuscript. YLT and GMZ managed the samples and analyzed the data, YLT, JHN, HLL and RZW managed the project samples and data. BHS reviewed the manuscript. XL designed the current study and reviewed the manuscript. All authors approved the final version of the manuscript. Acknowledgements The authors thank the Translational Medicine Research Center and the Hepatobiliary Research Institute (North Sichuan Medical College) for providing a research platform. Data Availability All data generated or analysed during this study are included in this published article. References George, L. F. et al. Ion Channel Contributions to Wing Development in Drosophila melanogaster. G3 (Bethesda) . 9 (4), 999–1008 (2019). Wu, S. et al. Disrupted potassium ion homeostasis in ciliary muscle in negative lens-induced myopia in Guinea pigs. Arch. Biochem. Biophys. ; 688 (108403). (2020). Crewther, S. G. et al. Ionic control of ocular growth and refractive change. Proc. Natl. Acad. Sci. U.S.A. 103 (42), 15663–15668 (2006). Zhang, X. & Hughes, B. A. KCNQ and KCNE potassium channel subunit expression in bovine retinal pigment epithelium. Exp. Eye Res. 116 , 424–432 (2013). Yang, Q. et al. 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Stepwise candidate drug screening for myopia control by using zebrafish, mouse, and Golden Syrian Hamster myopia models. EBioMedicine 65 , 103263 (2021). Munro, G., Erichsen, H. K. & Mirza, N. R. Pharmacological comparison of anticonvulsant drugs in animal models of persistent pain and anxiety. Neuropharmacology 53 (5), 609–618 (2007). Bialer, M. et al. Progress report on new antiepileptic drugs: a summary of the Ninth Eilat Conference (EILAT IX). Epilepsy research . ;83(1):1–43. (2009). Ohy, S. et al. Recent advances in therapeutic strategies that focus on the regulation of ion channel expression. Pharmacol. Ther. 160 , 11–43 (2016). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6127485","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":445192801,"identity":"f29ed6b9-35ce-4d00-a484-176a74a14af9","order_by":0,"name":"Qin Yang","email":"","orcid":"","institution":"Department of Ophthalmology of Affiliated Hospital, Medical School of Ophthalmology \u0026 Optometry, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Yang","suffix":""},{"id":445192802,"identity":"aaf4404b-2f4e-408b-b6c9-ef2542c72358","order_by":1,"name":"Jiahao Niu","email":"","orcid":"","institution":"Department of Ophthalmology of Affiliated Hospital, Medical School of Ophthalmology \u0026 Optometry, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jiahao","middleName":"","lastName":"Niu","suffix":""},{"id":445192803,"identity":"27f54ddb-f3a7-423b-beda-cee14c530a88","order_by":2,"name":"Yuling Tang","email":"","orcid":"","institution":"Department of Ophthalmology of Affiliated Hospital, Medical School of Ophthalmology \u0026 Optometry, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yuling","middleName":"","lastName":"Tang","suffix":""},{"id":445192804,"identity":"20f761f3-10a3-4deb-8897-181c3ca8f11d","order_by":3,"name":"Yuanyuan Hu","email":"","orcid":"","institution":"Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Hu","suffix":""},{"id":445192805,"identity":"4e36fb46-8bd2-4963-9bcd-71f4d0da2641","order_by":4,"name":"Guimei Zhou","email":"","orcid":"","institution":"Department of Ophthalmology of Affiliated Hospital, Medical School of Ophthalmology \u0026 Optometry, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Guimei","middleName":"","lastName":"Zhou","suffix":""},{"id":445192806,"identity":"7d96bbad-37f3-46d1-bf72-78cf1c412ce7","order_by":5,"name":"Huilan Liu","email":"","orcid":"","institution":"Department of Ophthalmology of Affiliated Hospital, Medical School of Ophthalmology \u0026 Optometry, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Huilan","middleName":"","lastName":"Liu","suffix":""},{"id":445192807,"identity":"59621b30-617d-4629-86e4-ae924931df3c","order_by":6,"name":"Runzhe Wang","email":"","orcid":"","institution":"Department of Ophthalmology of Affiliated Hospital, Medical School of Ophthalmology \u0026 Optometry, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Runzhe","middleName":"","lastName":"Wang","suffix":""},{"id":445192808,"identity":"beff48f3-143a-40a2-8091-33e17c95227d","order_by":7,"name":"Hongsheng Bi","email":"","orcid":"","institution":"Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hongsheng","middleName":"","lastName":"Bi","suffix":""},{"id":445192809,"identity":"7fe0569c-30ef-4cae-8bd7-02128d3a4ba4","order_by":8,"name":"Xuan Liao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDACdgjF2M/MfPgBcVqYoVpmtrOlGZCmZcN5HgUJonTwNzMffMxTc0d282EeBgOGGptoglokDrMlG8449sx422HeAw8YjqXlNhDUc5jHTOID2+HEbYf5EgwYGw4T1iJ/mP/7j4R/hxM3N/MYSBClxeAwDxvDx7bDiRuYidVieJjNWHJm32HjGYeBgZxAjF/kjjc//Mzz7bBsf//hww8+1NgQ4X0UkECa8lEwCkbBKBgFuAAA3qJAUKPnkWkAAAAASUVORK5CYII=","orcid":"","institution":"Department of Ophthalmology of Affiliated Hospital, Medical School of Ophthalmology \u0026 Optometry, North Sichuan Medical College","correspondingAuthor":true,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Liao","suffix":""}],"badges":[],"createdAt":"2025-02-28 10:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6127485/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6127485/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81098510,"identity":"9db24196-128c-4114-86a8-ba79ba82a486","added_by":"auto","created_at":"2025-04-22 08:20:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":215958,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of refraction (A, B) and AL (C, D) in each group at different time point. RTG, Retigabine; XE, XE991 (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6127485/v1/cdcfd7ee35534ce42b5262a9.png"},{"id":81097913,"identity":"bf9b80ec-fbfd-4e69-99d5-ac090c8b55d8","added_by":"auto","created_at":"2025-04-22 08:12:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":167256,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of IOP in each group at different injection time points. RTG, Retigabine; XE, XE991\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6127485/v1/df019e51046f0dcb5648b4cd.png"},{"id":81097910,"identity":"0c58e5b2-2c1b-434b-8e58-80fdb237dc91","added_by":"auto","created_at":"2025-04-22 08:12:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":100516,"visible":true,"origin":"","legend":"\u003cp\u003eThe mRNA expression of KCNQ5 in each group after 4-weeks FD. RTG, Retigabine; XE, XE991 (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6127485/v1/1eb9fa7469d2954aa6455d66.png"},{"id":81097917,"identity":"f2945d5f-393f-4278-9db6-a3bc138816d5","added_by":"auto","created_at":"2025-04-22 08:12:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":185285,"visible":true,"origin":"","legend":"\u003cp\u003eThe protein expression of KCNQ5 in each group after 4 weeks. (A) and (C) showed the western blot electrophoretic bands of each group. (B) and (D) showed the statistical results of gray level of the stripe. RTG, Retigabine; XE, XE991 (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6127485/v1/81e147c8cad6592e3ab19a6e.png"},{"id":81097914,"identity":"ddf9b646-5965-46ab-8071-68dc6354e4a6","added_by":"auto","created_at":"2025-04-22 08:12:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1272954,"visible":true,"origin":"","legend":"\u003cp\u003eCell apoptosis in each group after intravitreal injection. (A-E) showed the TUNEL staining results of each group; (F) showed the statistical results of the average optical density of the images. RTG, Retigabine; XE, XE991.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6127485/v1/8f7606f6f2e65f225682733b.png"},{"id":104072485,"identity":"c452cda0-b085-4a39-8527-69193165f75f","added_by":"auto","created_at":"2026-03-06 12:11:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2517757,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6127485/v1/1626c6f6-f573-421f-bb5b-4fa88801f78a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of KCNQ5 channel intervention on the development of form deprivation myopia in guinea pigs","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMyopia is the most common refractive error in clinical practice, which affects visual acuity and visual quality and even harms visual function. It is the main cause of visual impairment and the major public health problem worldwide.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Moreover, myopia among children and adolescents has become increasingly serious in recent years. However, at present, the pathogenesis of myopia has not been clarified, resulting in limited means of correction and a lack of effective intervention measures in clinic. Therefore, it is scientific and clinical significance to reveal the mechanism of myopia. Recent studies have shown that potassium ion (K\u003csup\u003e+\u003c/sup\u003e ) homeostasis is an important manifestation of myopia development, in which various related channels regulating potassium transport in ocular tissue may play a key role.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Our previous study revealed that the concentration of K\u003csup\u003e+\u003c/sup\u003e increased, while the expression of KCNQ5 was significantly down-regulated in the guinea pig retina of form-deprivation myopia (FDM). Studies have also found that KCNQ5 is expressed in bovine RPE and is involved in controlling the concentration of K\u003csup\u003e+\u003c/sup\u003e in the retina, which plays an important role in maintaining the physiological functions of photoreceptors, M\u0026uuml;ller cells and RPE cells.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Besides, we verified that the intervention effects of activating and inhibiting KCNQ5 channels on retinal muscarine (M) currents in guinea pigs through a series of in vitro experiments.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e These findings suggest that retinal KCNQ5 potassium channel may be involved in the occurrence and development of myopia. The present study aimed to observe the effect of KCNQ5 channel intervention on the development of FDM in guinea pigs through in vivo experiment, and to further investigate the role of KCNQ5 channel in myopia.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Study Design\u003c/h2\u003e \u003cp\u003eA total of 85 pigmented male guinea pigs aged three-week-old (120\u0026ndash;150 g) were supplied by Chongqing Tengxin Biotechnology Co., Ltd., The animals were raised at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃ and in a 12-h light-dark cycle with approximately 300\u0026ndash;500 lux.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Monocular FDM was induced by translucent facemasks made of latex balloons, the right eye was covered for a period of 4 weeks. All animal experiments were approved by the Animal Care and Ethics Committee of North Sichuan Medical College (NSMC 202116), and followed the Statement of Association for Research in Vision and Ophthalmology (ARVO) for the Use of Animals in Ophthalmic and Visual Research. All experimental procedures and related operations in this study were carried out in accordance with the standards of ARRIVE guidelines. The animals were randomly divided into 5 groups: control group (NC) with untreated bilateral eye, FD group with unilateral FDM, solvent group (FD\u0026thinsp;+\u0026thinsp;DMSO) with unilateral FDM and intraocular injection of a solvent containing 10% DMSO into the covered eye. Besides, on the basis of FD, retigabine group (FD\u0026thinsp;+\u0026thinsp;Retigabine) was injected with different concentrations of Retigabine, and XE991 group (FD\u0026thinsp;+\u0026thinsp;XE991) was injected with different concentrations of XE991. The concentrations of Retigabine were 100\u0026micro;M and 500\u0026micro;M, and XE991 were 20\u0026micro;M, 50\u0026micro;M and 100\u0026micro;M, respectively. The animals were then subdivided into 8 subgroups according to the above treatments and drug concentration differences (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe design of experimental grouping\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDrug concentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅠ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅡ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅢ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFD\u0026thinsp;+\u0026thinsp;DMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10% DMSO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅣ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFD\u0026thinsp;+\u0026thinsp;100 RTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u0026micro;M Retigabine\u0026thinsp;+\u0026thinsp;10% DMSO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅤ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFD\u0026thinsp;+\u0026thinsp;500 RTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u0026micro;M Retigabine\u0026thinsp;+\u0026thinsp;10% DMSO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFD\u0026thinsp;+\u0026thinsp;20 XE991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026micro;M XE991\u0026thinsp;+\u0026thinsp;10% DMSO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅦ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFD\u0026thinsp;+\u0026thinsp;50 XE991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u0026micro;M XE991\u0026thinsp;+\u0026thinsp;10% DMSO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅧ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFD\u0026thinsp;+\u0026thinsp;100 XE991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u0026micro;M XE991\u0026thinsp;+\u0026thinsp;10% DMSO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDrugs and Intravitreal injection\u003c/h3\u003e\n\u003cp\u003eDrugs Retigabine (selleckchem, United States) and XE991 (Sigma, United States) were dissolved in DMSO (Sigma, United States), and the solvent was prepared from 0.9% saline solution containing 10%DMSO. The drug stock solution was diluted with solvent before using to ensure that the DMSO content in each animal was maintained at 10%. Before injection, the diluted drugs were separately packed and stored in sterile EP tubes at 4℃. Injections were performed once every 3 days at a fixed time with the injection volume of 10\u0026micro;l. Iodophor was used to sterilize around the eyes, and aubucaine hydrochloride (Santen, Japan) were used for surface anesthesia. Oxyfloxacin ophthalmic ointment (Santen, Japan) was used twice daily to prevent infection. After four weeks, animals were euthanized and the ocular tissue was taken for follow-up studies.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eMeasurement of Ocular Biometric Parameters\u003c/h3\u003e\n\u003cp\u003eThe ocular biometric parameters were measured at five time points (0, 1, 2, 3, and 4 weeks of FD). Refraction was recorded by an independent experienced optometrist using retinoscopy (66 Vision-Tech Co., Ltd., China), and analyzed using the spherical equivalent (SE, SE\u0026thinsp;=\u0026thinsp;spherical power\u0026thinsp;+\u0026thinsp;1/2 cylindrical power).\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Guinea pigs were given cycloplegic drops (compound tropicamide; Santen Pharmaceutical Co., Ltd., Japan) at 5-min intervals for three times to dilate pupils and inhibit ocular accommodation. The anterior chamber depth, lens thickness, and vitreous cavity length (the sum of the three parameters was AL) (Zhang et al., 2018) were measured with an A-ultrasound (Cinescan, Quantel Medical, France) with an ultrasound probe frequency of 11 MHz, and the conduction velocities were set as 1557.5, 1723.3, and 1540 m/s, respectively. Before measurement, topical anesthesia was performed with oxybucaine hydrochloride (Santen Pharmaceutical Co., Ltd., Japan), and then the probe tip was perpendicular to the center of the cornea. Each measurement comprised an average of at least ten scans.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Intraocular pressure (IOP) was measured using a tonometer (iCare, Finland) during the same period after each intravitreal injection. The animals were measured under anaesthesia, and each eye was measured 5 times to take an average value for recording.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eTissue preparation\u003c/h3\u003e\n\u003cp\u003eAt the end of 4-week treatment, the guinea pigs were anesthetized with 3% sodium pentobarbital (50 mg/kg) until the cornea and pain reflex disappeared. The eyeballs were enucleated and positioned in a centrifuge tube and fixed in 4% paraformaldehyde for TUNEL staining. Additional eyeballs were enucleated, then the cornea, lens, and vitreous were quickly removed on ice. The retinas were snapped frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80◦C.\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eReal-time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was respectively extracted from the retinas using trizol reagent (Beyotime, Biotechnology, China) according to the manufacturer\u0026rsquo;s protocol. This was followed by reverse-transcription of 1 \u0026micro;g of total RNA to cDNA using Takara reverse transcriptase and the random primer provided in the kit (Takara, Japan). Real-time PCR was performed on a Roche LightCycler instrument with Takara SYBR green II kit using resultant cDNA as template. The primer sequences in this study were as follows: KCNQ5 forward primer 5'-ACTTGGCTGGGAAGGTTGCTTTC-3' and reverse primer 5'-GTGTTTCTGGCGGTGTTGTTCTTG-3\u0026rsquo;, β-actin forward primer 5'-CTGGGTATGGAATCCTGTGGCATC-3' and reverse primer 5'-CAGCACTGTGTTGGCATAGAGGTC-3'. The amplification was performed under the following cycling conditions: The first step was an initial denaturation at 95◦C for 30 s, followed by 40 cycles of denaturation at 95◦C for 10 s, annealing at 59◦C for 60 s, and extension at 97◦C for 1 s. At the end of the amplification, calculating the relative expression of target genes in each group with the 2^\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eThe RIPA lysis buffer (Beyotime Biotechnology, China) containing 1mM PMSF was added to the retina for full cracking to extract proteins from tissues. This was followed by the centrifugation at 12,000 rpm/min for 10 min to collect the supernatants for western blot analysis. Protein concentration was determined by the BCA kit (Beyotime Biotechnology, China). The samples were mixed with a 5 \u0026times; loading buffer (Beyotime Biotechnology, China) and then placed in 70◦C for 15 min. The protein samples were loaded on 8% SDS-PAGE gels and electrotransferred onto a nitrocellulose membrane. And the membranes were blocked with 5% non-fat milk at room temperature for 2h, the primary antibody of KCNQ5 (1:500, Invitrogen, United States) and β-tubulin (1:5,000, Beyotime Biotechnology, China) were incubated with the membranes at 4◦C overnight. After that, the membranes were incubated with goat anti-rabbit secondary antibody (1:10,000; Boster, China) at room temperature for 1 h. Finally, protein bands were visualized using the FUSION-FX7 imaging system (Vilber Lourmat, France) and the optical density was analyzed using Image J software.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTUNEL staining\u003c/h3\u003e\n\u003cp\u003eTUNEL staining (Kaiji Biotechnology Co. Ltd., China) was performed to detect apoptotic cells in the retina. The paraffin slices were baked overnight in an oven at 60℃. After dewaxing and rehydrating, the slices were washed 3 times with PBS for 5min each time. It was incubated with protease K solution at 37℃ for 30min (1:50). And then they were washed 3 times with PBS for 5min each time and incubated with TdT enzyme reaction solution at 37℃ for 1h away from light. After washing with PBS for 3 times, Streptavidin-HRP was incubated at 37℃ for 30min in the dark. After 3 washes in PBS, and then dropped with secondary antibody (Boster, China) and incubated at 37◦C for 1 h. The DAB reagent (ZSBg-Bio, China) was subsequently used to detect the immunoactivity. Then the sections were counterstained with hematoxylin (Boster, China), and dehydrated in different concentrations of ethanol. Finally, after the sections were sealed with neutral resin, images were captured using an optical microscope. Image J software was used to measure the optical density of positive retinal cells.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Deviation (SD), and Statistical software SPSS 25.0 was used for statistical analysis. GraphPad Prism 8.0 and Photoshop were performed for drawing. One-way analysis of variance was used to compare the differences in biological parameters and molecular levels among the groups, multiple comparisons between groups were performed by LSD-t method. All the results shown represent at least three independent experiments, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of drug intervention on refractive status and AL of guinea pigs\u003c/h2\u003e \u003cp\u003eThe results in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed that after 4 weeks, the refraction in the FD group was about \u0026minus;\u0026thinsp;3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99D and the AL was about 8.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11mm. Compared with the FD group, the refraction and AL of the eye in the FD\u0026thinsp;+\u0026thinsp;DMSO group had no statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.79, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.59, respectively). In FD\u0026thinsp;+\u0026thinsp;100\u0026micro;M Retigabine group and FD\u0026thinsp;+\u0026thinsp;500\u0026micro;M Retigabine group, both the refraction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively) and the AL (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) decreased. On the contrary, in FD\u0026thinsp;+\u0026thinsp;20\u0026micro;M XE991 group, FD\u0026thinsp;+\u0026thinsp;50\u0026micro;M XE991 group and FD\u0026thinsp;+\u0026thinsp;100\u0026micro;M XE991 group, the refraction increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.73, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively) and AL lengthened (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.82, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of drug intervention on intraocular pressure of guinea pig\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed the results of IOP in each group after each injection. There was no significant difference in IOP among all groups at each time point of intravitreal injection (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and the IOP fluctuated between 10-12mmHg in each group.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of drug intervention on mRNA level of KCNQ5 in guinea pig\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e showed the mRNA levels of KCNQ5 in retinal tissues of guinea pigs in each group. The mRNA expression level of FD and FD\u0026thinsp;+\u0026thinsp;DMSO groups was lower than the NC group. The expression levels of Retigabine at injection concentrations of 100\u0026micro;M and 500\u0026micro;M were significantly higher than the FD group, and the differences were statistically significant (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, the expression of KCNQ5 mRNA in the groups injected with XE991 gradually decreased with the increase of drug concentration, and when the concentration increased to 100\u0026micro;M, there was statistical significance in the expression of KCNQ5 mRNA compared with the FD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of drug intervention on protein level of KCNQ5 in guinea pig\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e showed that after the Retigabine was injected into the guinea pigs, the expression of KCNQ5 protein in retina was significantly up-regulated compared with FD group (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no significant difference in the expression of KCNQ5 protein between the FD\u0026thinsp;+\u0026thinsp;DMSO group and the low concentration (20\u0026micro;M) XE991 group, however, the expression of KCNQ5 protein in FD\u0026thinsp;+\u0026thinsp;50\u0026micro;M XE991 group and FD\u0026thinsp;+\u0026thinsp;100\u0026micro;M XE991 group was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of drug injection on apoptosis of guinea pig retina cells\u003c/h2\u003e \u003cp\u003eTUNEL staining showed that different degrees of apoptosis could be observed in the retina of NC group, FD group, injection solvent group (FD\u0026thinsp;+\u0026thinsp;DMSO), high concentration Retigabine group (500\u0026micro;M RTG) and high concentration XE991 group (FD\u0026thinsp;+\u0026thinsp;100\u0026micro;M XE991). Image J software analysis showed that the rate of positive cells in each group was 3.95% \u0026plusmn; 0.31% in NC group, 4.73% \u0026plusmn; 0.55% in FD group, 4.92% \u0026plusmn; 0.95% in FD\u0026thinsp;+\u0026thinsp;DMSO group, 4.81% \u0026plusmn; 0.48% in FD\u0026thinsp;+\u0026thinsp;500\u0026micro;M Retigabine group, and 5.13% \u0026plusmn; 1.01% in FD\u0026thinsp;+\u0026thinsp;100\u0026micro;M XE991 group, and there was no significant difference in TUNEL staining positive cell rate among all groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e transport is mainly regulated by K\u003csup\u003e+\u003c/sup\u003e channels and K\u003csup\u003e+\u003c/sup\u003e transporters. Previous studies have shown that K\u003csup\u003e+\u003c/sup\u003e transporters NKCC and Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e-ATPase are involved in the development of myopia.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e KCNQ5 is one of the important channels that may regulate K\u003csup\u003e+\u003c/sup\u003e transport between retina and choroid. The differential expression of KCNQ5 in myopic guinea pigs has been confirmed in the previous study, and the expression of KCNQ5 in mRNA and protein levels were lower than the NC group. Meanwhile, elevated K\u003csup\u003e+\u003c/sup\u003e concentration in the retina of FDM guinea pigs was also observed.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e These indicating that the KCNQ5 channel may be inhibited during the development of myopia, and preliminarily determined that it may be involved in the regulation mechanism of myopia.\u003c/p\u003e \u003cp\u003eKCNQ5 is a member of the KCNQ channel family, which is a voltage-dependent potassium channel, also known as the M channel, because it is sensitive to the regulation of Muscarinic acetylcholine receptors. Retigabine and XE991 act as specific activators and inhibitors of KCNQ channels respectively, and have also been widely used for positive and negative regulation of KCNQ5 channels.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e At present, most studies on KCNQ5 channel focus on electrophysiological and neurosystemic diseases, in which the changes of M-type potassium current regulated by KCNQ5 channel were explored.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e In the present study, it was found that the injection of activators and inhibitors of KCNQ5 channel into the guinea pig vitreous could regulate the expression of KCNQ5, and had different effect on the refractive state of guinea pigs, which proved that KCNQ5 channel was indeed involved in the development of FDM in guinea pigs.\u003c/p\u003e \u003cp\u003eAs a crucial regulator, M channels can inhibit neuronal excitability when activated, and vice versa. In fact, studies have found a link between the M channel and the myopia development. For example, atropine, as a Muscarinic acetylcholine receptor nonspecific antagonist, has been used clinically to intervene in myopia and has been shown to be effective. Animal experiments have also confirmed that acetylcholine antagonists, dopamine receptors and other related agents have a certain control effect on the occurrence and development of myopia.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e However, its side effects such as photophobia and paralytic regulation can be unpleasant, and even lead to cataracts.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Therefore, it is still significant to further explore myopia mechanism and potential drugs.\u003c/p\u003e \u003cp\u003eRetigabine plays a role in activating KCNQ2-5 channels. As a activator of KCNQ channels, and has been used as a specific activator of KCNQ5 channel in many studies.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Therefore, in this study, Retigabine was selected as an intervention drug of KCNQ5 channel for animal models. At the beginning of the study, it was found Retigabine could affect the development of refraction and AL when the concentration of the drug injected into the guinea pigs was increased to 100\u0026micro;M, and it was slower in FD\u0026thinsp;+\u0026thinsp;Retigabine group than in FD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). RT-PCR and Western Blot results showed that both low (100\u0026micro;M) and high (500\u0026micro;M) concentrations of Retigabine significantly increased the expression of KCNQ5, compared with FD group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This was consistent with the effect of Retigabine on refractive development. These results indicated that Retigabine can inhibit the development of myopia in guinea pigs by activating the expression of KCNQ5.\u003c/p\u003e \u003cp\u003eXE991 is a commonly drug for experimental studies on potassium channel inhibitors. \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e In this study, it was found that intra-abdominal injection of XE991 could cause a higher mortality in guinea pigs, which was speculated to be related to the related nerve response caused by XE991, but no further study was conducted. Therefore, in the follow-up experiment, we selected 3 concentrations as gradients to be administered the drug by intravitreal injection. The results showed that low concentration (20\u0026micro;M) of XE991 slightly inhibited the expression function of KCNQ5, but had no significant effect on refractive development (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05 ). However, the medium concentration (50\u0026micro;M) and high concentration (100\u0026micro;M) of XE991 can promote the development of refraction and AL of guinea pigs from the early modeling stage, and lead to higher transcription and protein expression levels of KCNQ5 than FD group. These suggested that XE991 as an inhibitor of KCNQ5 channels may play an opposite role in Retigabine, inhibiting the expression of KCNQ5 and promoting the development of FDM in guinea pigs.\u003c/p\u003e \u003cp\u003eThe administration route in this study was intravitreal injection, and the administration frequency was slightly higher (3 times/day). However, TUNEL staining results showed no statistically significant difference in the rate of positive cells among all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating that these drugs to intravitreal injection did not cause significant retinal toxicity. Therefore, to a certain extent, the drug concentration and administration method selected in this study were relatively safe. However, there are some limitations in our current study, including that mRNA and protein levels did not show dose-dependent activation and inhibition similar to the results of ocular parameters, possibly due to insufficient precision of drug concentration or differences of drug absorption in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, the present study underscores the significance of retinal KCNQ5 in myopia development in guinea pigs. Furthermore, it established that Retigabine exerts an inhibitory effect on the progression of FDM, while XE991 demonstrates an opposing influence. As previously acknowledged, we hope that our study will shed light on we aspire for our research to illuminate the pathogenesis of myopia and contribute to future prevention and treatment strategies for this condition.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by Natural Science Foundation Project of Science \u0026amp; Technology Department of Sichuan Province (No. 2023NSFSC0595), and Key Project of the Affiliated Hospital of North Sichuan Medical College (No. 2023ZD010).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQY performed the experiments, analyzed the data, and wrote the original manuscript. YLT and GMZ managed the samples and analyzed the data, YLT, JHN, HLL and RZW managed the project samples and data. BHS reviewed the manuscript. XL designed the current study and reviewed the manuscript. All authors approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors thank the Translational Medicine Research Center and the Hepatobiliary Research Institute (North Sichuan Medical College) for providing a research platform.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGeorge, L. F. et al. Ion Channel Contributions to Wing Development in Drosophila melanogaster. \u003cem\u003eG3 (Bethesda)\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e (4), 999\u0026ndash;1008 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, S. et al. 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Ther.\u003c/em\u003e \u003cb\u003e160\u003c/b\u003e, 11\u0026ndash;43 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"KCNQ5, Potassium channel, Retigabine, XE991, intravitreal injection","lastPublishedDoi":"10.21203/rs.3.rs-6127485/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6127485/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose: \u003c/strong\u003eTo investigate the effect of KCNQ5 on myopia and explore the mechanism by which potassium channel influence myopia in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eAnimals were divided into five groups: normal control, form deprivation (FD), solvent control, FD + Retigabine and FD + XE991. The concentrations of the Retigabine administered were 100μM and 500μM, while those for XE991 were 20μM, 50μM and 100μM. Injections and biometric examinations were performed at predetermined intervals. The expression levels were evaluated using RT-PCR and Western Blot, and cell apoptosis was detected via TUNEL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAfter four weeks, the progression of refraction and axial length in FD + Retigabine were significantly slowed down but accelerated in FD + XE991 compared to the FD. Statistically significant differences were observed between 50μM and 100μM. There was no significant difference in IOP among groups. The mRNA and protein levels of KCNQ5 increased in FD + Retigabine but decreased significantly in doses of XE991 treatment when compared with FD controls. TUNEL staining indicated no significant differences in positive cell rates across all retinal layers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eIntervention targeting KCNQ5 channels appears to modulate the development of FD myopia, suggesting that potassium channel may play a role in underlying mechanisms associated with myopia.\u003c/p\u003e","manuscriptTitle":"The effect of KCNQ5 channel intervention on the development of form deprivation myopia in guinea pigs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 08:12:29","doi":"10.21203/rs.3.rs-6127485/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"30ea9f82-47c0-4230-8737-b589f45cbfa9","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47398405,"name":"Biological sciences/Biochemistry"},{"id":47398406,"name":"Biological sciences/Physiology"},{"id":47398407,"name":"Health sciences/Health care"},{"id":47398408,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2026-03-06T12:10:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-22 08:12:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6127485","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6127485","identity":"rs-6127485","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}